U.S. patent number 9,056,097 [Application Number 13/010,473] was granted by the patent office on 2015-06-16 for composite of amorphous calcium phosphate/calcium sulfate hemihydrate (csh/acp) for bone implantation and process for producing the same.
This patent grant is currently assigned to Taipei Medical University. The grantee listed for this patent is Wei-Jen Chang, Haw-Ming Huang, Dian-Yu Ji, Sheng-Yang Lee, Malosi Poma, Duen-Cheng Wang, Hong-Da Wu, Jen-Chang Yang. Invention is credited to Wei-Jen Chang, Haw-Ming Huang, Dian-Yu Ji, Sheng-Yang Lee, Malosi Poma, Duen-Cheng Wang, Hong-Da Wu, Jen-Chang Yang.
United States Patent |
9,056,097 |
Yang , et al. |
June 16, 2015 |
Composite of amorphous calcium phosphate/calcium sulfate
hemihydrate (CSH/ACP) for bone implantation and process for
producing the same
Abstract
The invention provides a composite of .alpha.-calcium sulfate
(CS) hemihydrate/amorphous calcium phosphate (.alpha.-CSH/ACP),
comprising .alpha.-CSH and ACP at a weight ratio of about 10:90 to
about 90:10. Particularly, the composite of the invention has a
resorption period of 3-6 months. The invention also provides a
one-pot process for producing .alpha.-CSH/ACP composite of the
invention. The one-pot process of the invention can produce
.alpha.-CSH and ACP in a single process and easily obtain
.alpha.-CSH/ACP composite.
Inventors: |
Yang; Jen-Chang (Taipei,
TW), Lee; Sheng-Yang (Taipei, TW), Wang;
Duen-Cheng (Taipei, TW), Huang; Haw-Ming (Taipei,
TW), Chang; Wei-Jen (Taipei, TW), Poma;
Malosi (Taipei, TW), Wu; Hong-Da (Taipei,
TW), Ji; Dian-Yu (Taipei, TW) |
Applicant: |
Name |
City |
State |
Country |
Type |
Yang; Jen-Chang
Lee; Sheng-Yang
Wang; Duen-Cheng
Huang; Haw-Ming
Chang; Wei-Jen
Poma; Malosi
Wu; Hong-Da
Ji; Dian-Yu |
Taipei
Taipei
Taipei
Taipei
Taipei
Taipei
Taipei
Taipei |
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A |
TW
TW
TW
TW
TW
TW
TW
TW |
|
|
Assignee: |
Taipei Medical University
(Taipei, TW)
|
Family
ID: |
46544344 |
Appl.
No.: |
13/010,473 |
Filed: |
January 20, 2011 |
Prior Publication Data
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|
Document
Identifier |
Publication Date |
|
US 20120189683 A1 |
Jul 26, 2012 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
19/08 (20180101); A61L 27/425 (20130101); A61L
27/427 (20130101); A61K 33/42 (20130101) |
Current International
Class: |
A61K
33/42 (20060101); A61L 27/42 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2005084726 |
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Sep 2005 |
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CH |
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Other References
Nillson et al. (J Mater Sci: Materials in Medicine 2003, 14,
399-404). cited by examiner .
Nilsson et al. Nilsson et al. (J Biomed Mater Res 2002;61:600-607).
cited by examiner .
A Novel Resorbable Composite of Calcium Sulfate and Amorphous
Calcium Phosphate Bone Substitute for Dental Implants, Abstract of
Master Thesis of Malosi Poma (English abstract provided), published
on line on Jul. 21, 2010. cited by applicant .
Kawai T, Murakami S, Hiranuma H, Sakuda M.: "Healing after removal
of benign cysts and tumors of the jaws. A radiologic appraisal,"
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995; 79(4):
517-25. cited by applicant .
Kawai T, Murakami S, Hiranuma H, Sakuda M.: "Radiographic changes
during bone healing after mandibular fractures," Br J Oral
Maxillofac Surg 1997; 35(5): 312-8. cited by applicant .
Urban RM, Turner TM, Hall DJ, Inoue N, Gitelis S.: "Increased bone
formation using calcium sulfate-calcium phosphate composite graft,"
Clin Orthop Relat Res 2007;459:110-7. cited by applicant .
Nilsson M, Wang JS, Wielanek L, Tanner KE, Lidgren L.:
"Biodegradation and biocompatability of a calcium
sulphate-hydroxyapatite bone substitute," J Bone Joint Surg
2004;86(1):120-125. cited by applicant .
Osteoset(R) T, Medicated Bone Graft Substitute, Technical
Monograph, Wright Medical Technologies, Inc., 2006, pp. 1-15. cited
by applicant.
|
Primary Examiner: Arnold; Ernst V
Attorney, Agent or Firm: McDonnell Boehnen Hulbert &
Berghoff LLP
Claims
What is claimed is:
1. A composite of .alpha.-calcium sulfate (CS)
hemihydrate/amorphous calcium phosphate (.alpha.-CSH/ACP), which
comprises .alpha.-CSH and ACP at a weight ratio of about 40:60 to
about 70:30.
2. The composite of claim 1, wherein the .alpha.-CSH is
.alpha.-CaSO.sub.4. 0.5H.sub.2O.
3. The composite of claim 1, wherein the .alpha.-CSH and ACP is at
a weight ratio of about 40:60, about 50:50, about 60:40, or about
70:30.
4. The composite of claim 1, which is in granular form.
5. The composite of claim 1, which is in powder form.
6. The composite of claim 1, where is in paste form.
7. A process for producing .alpha.-CSH/ACP composite, which
comprises the following steps: (a) separately dissolving a calcium
ion-containing solution and a sulfate ion containing solution in
calcium chloride solutions; (b) heating the solutions of (a) to a
temperature of about 80.degree. C. to 100.degree. C.; and mixing
the two solutions for reaction to produce .alpha.-calcium sulfate
hemihydrate (.alpha.-CSH); (c) filtrating the resulting solution of
(b) to separate .alpha.-CSH solid from the solution; (d) adding a
phosphate compound to the solution in (c) to react with calcium
chloride in (a) to obtain amorphous calcium phosphate (ACP); and
(e) mixing .alpha.-CSH of (c) and ACP of (d) and then adding water
to produce a composite of .alpha.-calcium sulfate hemihydrate and
amorphous calcium phosphate (.alpha.-CSH/ACP) at a .alpha.-CSH/ACP
weight ratio of about 40:60 to about 70:30.
8. The process of claim 7, wherein the calcium ion-containing
solution is a solution containing calcium chloride, calcium
hydroxide, calcium nitrate or calcium oxide.
9. The process of claim 7, wherein the calcium ion-containing
solution is calcium nitrate solution.
10. The process of claim 7, wherein the sulfate ion-containing
solution is a solution containing potassium sulfate or sulfuric
acid.
11. The process of claim 7, wherein in step (d), the phosphate
compound is sodium dihydrogen phosphate (Na.sub.2HPO.sub.3),
potassium dihydrogen phosphate (K.sub.2HPO.sub.3) or phosphoric
acid (H.sub.3PO4).
12. The process of claim 7, wherein in step (d), the phosphate
compound is sodium dihydrogen phosphate.
13. The process of claim 7, wherein the reaction in step (d) is
performed at a basic pH.
14. The process of claim 13, wherein the pH value ranges from 7.5
to 10.0.
15. The process of claim 7, wherein the weight ratio of .alpha.-CSH
and ACP is about 40:60, about 50:50, about 60:40, or about
70:30.
16. The process of claim 7, wherein in step (e), the .alpha.-CSH is
.alpha.-CaSO.sub.4.0.5H.sub.2O.
Description
FIELD OF THE INVENTION
The invention provides a composite of .alpha.-calcium sulfate (CS)
hemihydrate/amorphous calcium phosphate (.alpha.-CSH/ACP) and a
process for production thereof. Particularly, the composite of the
invention comprises .alpha.-CSH and ACP at a weight ratio of about
10:90 to about 90:10.
BACKGROUND OF THE INVENTION
Due to concerns over potential immune response and supply shortages
associated with autografts and allografts, synthetic bone graft
substitutes (SBGSs) for augmenting the bone have rapidly gained
popularity in the field of implantation. SBGSs are widely used in
implantation due to their biocompatibility, osteoconduction, and
minimal risk of disease transmission. Typical ceramic bone graft
materials such as hydroxyapatite (HA,
Ca.sub.10(PO.sub.4).sub.6(OH).sub.2), .beta.-tricalcium phosphate
(.beta.-TCP, .beta.-Ca.sub.3(PO.sub.4).sub.2) and calcium sulfate
(CS, CaSO.sub.4), can be presented in different product forms such
as powder, granule, pellet, putty, or block to apply to various
bone damage conditions. At present, many fabrication methods for
bone grafts have been developed, as summarized in Table 1.
TABLE-US-00001 TABLE 1 Product name (producer, factory) Composition
(content) Comments (phase) Healos (Depuy Spine) Sponge ProOsteon
(Interpore Int., Particulate or block brittle. USA) Previous name:
Replam Radiopaore size 190-230 .mu.m) Hydroxyapatite-Porites or
500: Porites Gonipora (large RHAP pores) R: Resorbcity impedes
assessment of healing. Slow resorption R-form Collagraft (Zimmer
Inc, USA) HA coated 70% Type I bovine Granules and strips require
collagen augmentation with aspirated marrow MBCP (Biomatlante)
Replaniform coralline Granules, rectangular sticks, macroporous HA
200: Porites cylinders or wedges (pable Triosite (Zimmer Europe
Ltd, 60% HA, 40% TCP Also called MBCP UK) (macroporous biphasic
calcium phosphate) or BCP BCP (Bioland) 60% HA, 40% TCP Ostilit
(Stryker Howmedica 20% HA, 80% TCP, without Granules and blocks for
Osteonics, UK) macroporous nonstructural grafts BoneSave (Stryker
20% HA, 80% TCP, Granules, stronger than Ostilit, Howmedica
Osteonics, UK) pore size: 400-600 .mu.m for use as a void filler
and in grafting Cerasorb ORTHO (curasan) Pure phase .beta.-TCP,
Granular size being 500-1,000 .mu.m micropores: <80 .mu.m or
1,000-2,000 .mu.m Vitoss .TM. Scaffold (curasan) .beta.-TCP,
micropores: <1-1000 .mu.m Morsel (1-4 mm sizes) and blocks (9
.times. 23 mm cylinder) Conduit .TM. TCP Granules >99%
(.beta.-TCP) Ca.sub.3(PO.sub.4).sub.2, Irregular shaped granules
(DePuy Spine) pore: 1-600 .mu.m having an average diameter between
1.5 and 3 mm Cellplex .TM. TCP synthetic Porous calcium phosphate
cancellous bone (Wright) made from TCP, pore size: 100-400 .mu.m
Ceros 82 .beta.-TCP, porosity varies to Lower compressive strength
adjust resorption between 6 than Ceros 80 and 12 months Synthes
(USA) chronOS .TM. .beta.-TCP pore size: 100-500 .mu.m Granules,
blocks, wedge and (Synthes) cylinders Calciresorb (Ceraver Osteal,
Porous TCP Periodontal applications France) Synthograf (Milter,
USA) Small size and dense TCP Periodontal applications Augmen
(Milter, USA) Large size and dense TCP Periodontal applications
Skelite .TM. (Millenium Multiphase, porous calcium Granules and
blocks Biologix) phosphate Norian Skeletal Repair System
Self-setting calcium phosphate Injectable cement, (SRS) cement
augmentation of fracture
There is great demand in the field of implementation surgery for an
ideal SBGS. The in vivo resorption rate is one vital property of
SBGSs that can be improved. SBGSs for implants should be rapidly
resorbable and replaced by new bone so that implants can be placed
as early as possible in the augmented site. However, the ideal
resorption period for SBGS use in implants (especially in
dentistry) is still uncertain. Clinical studies have reported that
a stress-free healing period of 3-6 months is prerequisite to
implant osseointegration. For example, a 3-6-month healing period
was proposed by Kawai et al. in their radiographic studies of
healing of jawbone defects and fractures (Kawai T, Murakami S,
Hiranuma H, Sakuda M. Healing after removal of benign cysts and
tumors of the jaws. A radiologic appraisal. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod 1995; 79(4): 517-25; Kawai T, Murakami S,
Hiranuma H, Sakuda M. Radiographic changes during bone healing
after mandibular fractures. Br J Oral Maxillofac Surg 1997; 35(5):
312-8). It was suggested that at a natural healing rate, most
jawbone defects required an average time of 3-6 months to heal.
Calcium sulfate (CS) is a rapidly resorbable and biocompatible bone
substitute with a bone regeneration effect and angiogenic effect.
Depending on the amount of crystal water, calcium sulfate can be
classified as calcium sulfate dihydrate (CaSO.sub.4.2H.sub.2O,
i.e., gypsum), calcium sulfate hemihydrate (CaSO.sub.4.0.5H.sub.2O,
i.e., plaster of Paris) or calcium sulfate anhydrite (CaSO.sub.4).
The following are the chemical reaction formulae of the above
reactions: Dehydration:
CaSO.sub.4.2H.sub.2O(s)+heat.fwdarw.CaSO.sub.4.1/2H.sub.2O(s)+11/2H.sub.2-
O CaSO.sub.4.1/2H.sub.2O(s)+heat.fwdarw.CaSO.sub.4+2H.sub.2O
Hydration:
CaSO.sub.4.1/2H.sub.2O(s)+3/2H.sub.2O.fwdarw.CaSO.sub.4.2H.sub.2O(s)
The in vivo resorption period of calcium sulfate hemihydrate is
longer than that of calcium sulfate dihydrate. However, calcium
sulfate hemihydrate will transform into calcium sulfate dihydrate
through hydration. Therefore, in commercial processes for producing
tablet calcium sulfate bone substitute, water is added to calcium
sulfate hemihydrate to form tablet calcium sulfate dihydrate and
then the tablet calcium sulfate dihydrate is transformed into
calcium sulfate hemihydrate by dehydration. CS is associated with
many other biomaterials. In vitro studies of the attachment of
osteoblast cells to CS and the resorption of CS by osteoclasts have
been reported. However the high in vivo resorption rate of CS of
1-2 months is considered too rapid, as it can limit bone
regeneration and cause serous drainage in some clinical
applications.
It has been recommended that merging CS with a less-resorbable
calcium phosphate compound would be better for human applications.
Many studies have worked on combining CS with other calcium
phosphates like HAp, .beta.-tricalcium phosphate (.beta.-TCP), and
.alpha.-tricalcium phosphate (.alpha.-TCP) (Urban R M, Turner T M,
Hall D J, Inoue N, Gitelis S. Increased bone formation using
calcium sulfate-calcium phosphate composite graft. Clin Orthop Mat
Res 2007; 459:110-7; Nilsson M, Wang J S, Wielanek L, Tanner K E,
Lidgren L. Biodegradation and biocompatability of a calcium
sulphate-hydroxyapatite bone substitute. J Bone Joint Surg 2004;
86(1):120-125). US Publication No. 20050119746 provides an
artificial bone mineral substitute material comprising at least one
ceramic and at least one water soluble non-ionic X-ray contrast
agent and illustrates an embodiment comprising 1-30% calcium
sulfate hemihydrate and 50-99% .alpha.-TCP. However, their
resorption rates were far too slow.
Amorphous calcium phosphate (ACP; with an approximate compositional
formula of Ca.sub.3(PO.sub.4).sub.2.0.8H.sub.2O) is a
non-crystalline and the most soluble form of tricalcium phosphate.
It is usually treated in vivo as a precursor of biological bone
apatite during bone formation. In vitro, ACP is the first phase
that precipitates from a supersaturated solution prepared by
rapidly mixing solutions containing calcium and phosphate ions. The
composition and poor crystalline structure of ACP mimic natural
bone apatite, and would be a better bone substitute than highly
crystalline hydroxyapatite (HAp). Previous studies confirmed that
ACP shows better bioactivity than HAp, because greater adhesion,
proliferation, and differentiation of osteogenic cells were
observed on ACP substrates than crystalline HAp substrates.
However, ACP will transform to Hap with longer resorption rate
after contacting water through phase transformation.
U.S. Pat. No. 7,351,280 relates to a composition and method for
producing interconnective macroporous, resorbable and injectable
calcium phosphate-based cements (MICPCs), which provide a
self-setting calcium phosphate cement (CPC). The invention of the
patent adds carbonate, magnesium, zinc, fluoride and pyrophosphate
ions to stabilize ACP. U.S. Pat. No. 7,670,419 discloses a
hydraulic cement based on calcium phosphate for surgical use
comprising A) a first component comprising powder particles of
calcium phosphate; and B) a second component comprising water. The
invention of the patent uses ACP that is preheated to 500.degree.
C. and then milled to shorten time of hydrating and hardening CPC.
US Publication No. 20020183417 relates to a calcium phosphate bone
graft material, to a process for making the calcium phosphate bone
graft material, and to an osteoimplant fabricated from the calcium
phosphate bone graft material. The invention uses plasma spray to
coat ACP on the surface of Hap to form a new bone substitute. US
Publication No. 20080014242 discloses a synthetic bone substitute
material suitable for use as a replacement for cancellous bone in a
bone graft composition, the material comprising a reticulated
framework of interconnecting bioceramic struts defining an
interconnecting interstitial void volume, and a solid non-porous
composition substantially filling the interstitial void volume and
in intimate contact with the reticulated framework, the
pore-filling composition comprising calcium sulfate. However, the
bone substitutes provided in the above prior art cannot provide
satisfactory resorption rate.
Therefore, there remains a need in the art for improved bone
substitute materials providing resorption rate suitably parallel to
the natural healing rate of the human bone.
SUMMARY OF THE INVENTION
The invention provides a composite of .alpha.-calcium sulfate
hemihydrate/amorphous calcium phosphate (.alpha.-CSH/ACP), which
comprises .alpha.-CSH and ACP at a weight ratio of about 10:90 to
about 90:10.
The invention also provides a process for producing .alpha.-CSH/ACP
composite, which comprises the following steps: (a) separately
dissolving a calcium ion-containing solution and a sulfate
ion-containing solution in calcium chloride solutions; (b) heating
the solutions of (a) to a temperature of about 80.degree. C. to
100.degree. C.; and mixing the two solutions for reaction to
produce .alpha.-calcium sulfate hemihydrate (.alpha.-CSH); (c)
filtrating the resulting solution of (b) to separate .alpha.-CSH
solid from the solution; (d) adding a phosphate compound to the
solution in (c) to react with calcium chloride in (a) to obtain
amorphous calcium phosphate (ACP); and (e) mixing .alpha.-CSH of
(c) and ACP of (d) and then adding water to produce a composite of
.alpha.-calcium sulfate hemihydrate and amorphous calcium phosphate
(.alpha.-CSH/ACP).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(a) shows the SEM graph of .alpha.-CSH; FIG. 1(b) shows the
SEM graph of ACP; FIG. 1(c) shows the X-ray diffraction (XRD)
pattern of .alpha.-CSH and ACP; and FIG. 1(d) shows the DSC
thermogram of .alpha.-CSH.
FIG. 2 shows X-ray diffraction (XRD) pattern of transformed ACP,
.alpha.-CSH/ACP, CSH and CSD.
FIG. 3 shows the SEM graph of .alpha.-CSH/ACP of the invention.
FIG. 4(a) shows the graph of the dissolution test for calcium
sulfate (CS) alone and composite .alpha.-CSH/ACP (60/40) in PBS,
and FIG. 4(b) shows XRD diagram of CS and .alpha.-CSH/ACP residues
after the dissolution test in PBS.
FIG. 5 shows the histological micrographs of bone defects filled
with .alpha.-CSH/ACP (60/40), Osteoset.RTM. and an empty defect
(Original magnification of 20.times. for all images; CT, connective
tissue; NB, new bone).
FIG. 6 shows extrapolation graph of the complete healing period for
bone defects in the canine model treated with .alpha.-CSH/ACP
(60/40) bone substitutes.
DETAILED DESCRIPTION OF THE INVENTION
The invention develops a resorbable composite of .alpha.-calcium
sulfate (CS) hemihydrate/amorphous calcium phosphate
(.alpha.-CSH/ACP) for use as bone graft substitute, and a one-pot
process for manufacture of a resorbable bone graft substitute of
.alpha.-calcium sulfate (CS) hemihydrate/amorphous calcium
phosphate (.alpha.-CSH/ACP). Unexpectedly, the .alpha.-CSH/ACP
composite of the invention provides a resorption period of 3-6
months that is specifically suitable for dental implantation
surgery. Moreover, the one-pot process of the invention can produce
.alpha.-CSH and ACP in a single process and easily obtain
.alpha.-CSH/ACP composite.
In one aspect, the invention provides a composite of
.alpha.-calcium sulfate hemihydrate/amorphous calcium phosphate
(.alpha.-CSH/ACP), which comprises .alpha.-CSH and ACP at a weight
ratio of about 10:90 to about 90:10. In one embodiment, the
.alpha.-CSH is .alpha.-CaSO.sub.4.0.5H.sub.2O. In another
embodiment of the invention, the .alpha.-CSH and ACP is at a weight
ratio of about 10:90, about 20:80, about 30:70, about 40:60, about
50:50, about 60:40, about 70:30, about 80:20 or about 90:10. In a
further embodiment of the invention, the -CSH/ACP of the invention
is in granular form, powder form or paste form.
According to the invention, the natural healing rate of the human
bone should parallel the ideal resorption rate of synthetic bone
graft substitute (SBGS) for implants. The invention unexpectedly
found that .alpha.-CSH and ACP at a specific ratio will generate
hydration by adding water, whereas .alpha.-CSH and ACP will not
transform into calcium sulfate dehydrate (CSD) and Hap, which needs
longer time for resorption respectively. The invention combines
.alpha.-CSH with ACP to create a biocompatible SBGS that could be
resorbed and substituted by bone within a 3-6-month period, and
this resulting .alpha.-CSH/ACP composite can mimic the natural bone
regeneration rate with a 3-6 month period. Preferably, the natural
bone is jaw bone, so the composite of the invention is especially
suitable for dental implantation.
According to the invention, the addition of ACP to CS improves the
osteoconductivity of the composite by reducing the resorption rate
and mimicking the natural bone structure and mineral components.
The .alpha.-CSH/ACP bone substitutes of the invention were
biocompatible, osteoconductive, and resorbable with a
mathematically estimated resorption period of about 3-6 months. The
novel composite CSH/ACP of the invention can shorten the implant
healing and treatment periods. The matching of resorption rates of
synthetic bone graft substitutes (SBGS) with the healing rate for
extraction sockets is highly desirable for implants. Therefore, the
composite of the invention is an ideal SBGS for implants.
In another aspect, the invention provides a process for producing
.alpha.-CSH/ACP composite, which comprises the following steps: (a)
separately dissolving a calcium ion-containing solution and a
sulfate ion-containing solution in calcium chloride solutions; (b)
heating the solutions of (a) to a temperature of about 80.degree.
C. to 100.degree. C.; and mixing the two solutions for reaction to
produce .alpha.-calcium sulfate hemihydrate (.alpha.-CSH); (c)
filtrating the resulting solution of (b) to separate .alpha.-CSH
solid from the solution; (d) adding a phosphate compound to the
solution in (c) to react with calcium chloride in (a) to obtain
amorphous calcium phosphate (ACP); and (e) mixing .alpha.-CSH of
(c) and ACP of (d) and then adding water to produce a composite of
.alpha.-calcium sulfate hemihydrate and amorphous calcium phosphate
(.alpha.-CSH/ACP).
The invention provides a one-pot process for producing the
.alpha.-CSH/ACP composite of the invention. The process of the
invention can produce .alpha.-CSH and ACP simultaneously and obtain
the .alpha.-CSH/ACP composite by mixing the resulting .alpha.-CSH
and ACP. Briefly, the above reactions can be accomplished in a
one-pot process and .alpha.-CSH, ACP and .alpha.-CSH/ACP can be
easily produced through the one-pot process.
According to the invention, in step (a) of the process, a calcium
ion-containing solution and a sulfate ion-containing solution are
separately solved in calcium chloride solutions. In an embodiment
of the invention, the calcium ion-containing solution is, but is
not limited to, a solution containing calcium chloride, calcium
hydroxide, calcium nitrate or calcium oxide. Preferably, the
calcium ion-containing solution is calcium nitrate solution. In
another embodiment of the invention, the sulfate ion-containing
solution is, but is not limited to, a solution containing potassium
sulfate or sulfuric acid. According to the invention, the calcium
chloride in the process is used as crystallization catalyst to
insure that the crystal phase of calcium sulfate hemihydrate
produced in step (b) will not transform into calcium sulfate
dihydrate.
According to the invention, in step (b) of the process, the
solutions of (a) are heated to a temperature higher than 80.degree.
C. and then mixed for reaction to produce .alpha.-calcium sulfate
hemihydrate (.alpha.-CSH). Preferably, the solutions are heated to
a temperature of 95.degree. C. According one embodiment of the
invention, the reaction time is at least 2 hours.
According to the invention, in step (c) of the process, the
resulting solution of (b) is subjected to filtration to separate
.alpha.-CSH solid from the solution, whereby .alpha.-CSH and liquid
part can be obtained respectively.
According to the invention, in step (d) of the process, a phosphate
compound is added to the solution in (c) so that it reacts with
calcium chloride in (a) to obtain amorphous calcium phosphate
(ACP). In an embodiment of the invention, the phosphate compound
is, but is not limited to, sodium dihydrogen phosphate
(Na.sub.2HPO.sub.3), potassium dihydrogen phosphate
(K.sub.2HPO.sub.3) or phosphoric acid (H.sub.3PO4). Preferably, the
phosphate compound is sodium dihydrogen phosphate. In another
embodiment of the invention, the reaction is performed at a basic
pH. Preferably, the pH value ranges from 7.5 to 10.0.
According to the invention, in step (e) of the process, .alpha.-CSH
of (c) and ACP of (d) are mixed to form a mixture. Subsequently,
water is added to the mixture to produce a composite of
.alpha.-calcium sulfate hemihydrate and amorphous calcium phosphate
(.alpha.-CSH/ACP). In one embodiment of the invention, the
.alpha.-CSH and ACP is mixed at a weight ratio of about 10:90 about
90:10. According to the invention, the addition of water to the
mixture of .alpha.-CSH and ACP will generate hydration reaction to
produce and harden the composite of .alpha.-CSH/ACP. In one
embodiment of the invention, the .alpha.-CSH and ACP are mixed at a
specific ratio range to have desired resorption period. Preferably,
the .alpha.-CSH is .alpha.-CaSO.sub.4.0.5H.sub.2O. In another
embodiment, the weight ratio of .alpha.-CSH and ACP is about 60:40.
Preferably, the .alpha.-CSH/ACP composite of the invention provides
a resorption period of 3-6 months.
EXAMPLES
Example 1
Preparation of .alpha.-CSH/ACP Composite of the Invention
Preparation of .alpha.-CSH
Pure .alpha.-CSH was prepared by a wet precipitation method using
calcium chloride (CaCl.sub.2) as a crystallization catalyst.
Calcium nitrate (Ca(NO.sub.3).sub.24H.sub.2O) of 0.1 mole and 0.1 M
potassium sulfate (K.sub.2SO.sub.4) were separately dissolved in 50
ml of a 3.5 M CaCl.sub.2 solution. Both solutions were preheated to
95.degree. C.; then they were mixed and incubated at atmospheric
pressure for 2 h. The detailed procedures are reported in U.S. Pat.
No. 7,700,066. The crystalline phase of the harvested .alpha.-CSH
was identified by x-ray diffraction (XRD), differential scanning
calorimetric (DSC), and scanning electron microscopy (SEM)
analysis. The crystal morphology of the .alpha.-CSH was examined by
SEM and is shown in FIG. 1a. The texture of CS revealed relatively
thin, long, needle-type crystals that interlocked with each other.
Additionally, XRD analysis shown in FIG. 1c displayed typical
2-theta (0) values of the peaks at 14.75.degree., 25.71.degree.,
29.76.degree., and 31.91.degree. associated with the crystal planes
of (110), (310), (220), and (-114), indicating the character of
.alpha.-CSH. The DSC thermogram (FIG. 1d) showed an endothermic
peak at approximately 210.degree. C. and a small exothermic peak at
230.degree. C. demonstrating the characteristics of
.alpha.-CSH.
Preparation of ACP
A buffering medium at pH 9 was prepared by the addition of sodium
hydroxide (NaOH) into 100 ml of distilled water. The preparation of
ACP was conducted by rapidly adding 100 ml of a sodium phosphate
dibasic (J.T. Baker, ST, USA) aqueous solution (2.33 M) and 100 ml
of a CaCl.sub.2 solution (3.50 M) to the buffering medium. The
envisaged ACP was filtered by high-power filtration (Sibata,
Circulating Aspirator WJ-20, Tokyo, Japan) and stored in a freezer.
It was freeze-dried for 2 days, then its crystalline structure was
confirmed by XRD analysis. The XRD pattern of the calcium phosphate
prepared by the wet precipation method disclosed a broad peak
characteristic for the amorphous structure (FIG. 10. SEM
photographs in FIG. 1b display flaky particles of ACP.
Preparation of .alpha.-CSH/ACP (60/40) Granules
.alpha.-CSH and ACP were mixed together (in a 60:40 weight ratio)
with deionized water at a powder to liquid ratio of 10:6. The
composite was then allowed to set at room temperature before being
dried in an oven at 80.degree. C. Using a pestle, the material was
ground in a grinding bowl and sieved to isolate particles of
500-840 .mu.m. After mixing .alpha.-CSH with water at a
powder-to-liquid ratio of 10:6 for 24 h, XRD analysis revealed
complete transformation of .alpha.-CSH to new diffraction peaks
located at 11.64.degree., 20.75.degree., 23.41.degree., and
29.14.degree. correlated with the characteristic crystal planes of
(020), (021), (040) and (041) for CSD after the hydration reaction.
In contrast, when .alpha.-CSH/ACP (60/40) was mixed with water
overnight, the hydrated composite displayed XRD diffraction peaks
for CSH only located at 14.75.degree., 25.66.degree.,
29.76.degree., and 31.91.degree. which were associated with the
crystal planes of (110), (310), (220), and (-114) with no
detectable peaks indicating the phase transformation to CSD (FIG.
2). FIG. 3 shows the SEM of the .alpha.-CSH/ACP (60/40).
Example 2
In Vitro Dissolution Test of .alpha.-CSH/ACP
In vitro dissolution tests are essential to demonstrate equivalence
in dissolution behavior for resorbable biomaterials in clinical
use. .alpha.-CSH/ACP (60/40) and CS weighing 1 g for each test
material were prepared. Samples were placed in plastic tea bags,
put in polyethylene tubes, and weighed to an accuracy of 0.01 mg.
Subsequently 50 ml of phosphate-buffered solution (PBS) were added
to each tube and incubated in a shaking bath (B603D, FIRSTREK, ST,
USA) at 37.degree. C. and shaken at a speed of 30 rpm. After 3
days, the polythene tubes were centrifuged (Hermle, Z 323 K, United
Corps, Germany), and the PBS was carefully removed. Samples were
washed with 30 ml of deionized water, dried, and cooled before
their new weight was recorded. Another 50 ml of fresh PBS was
added, and the samples were added to the shaking bath for another 3
days. Dissolution rates of samples were measured by calculating the
percentages of sample weight retention. The experiment was
discontinued after 3 months when insignificant percentage weight
loss was detected. FIG. 4a reveals the weight-retention profiles
for .alpha.-CSH/ACP (60/40) and CS at various time periods up to 90
days in PBS. Both .alpha.-CSH/ACP (60/40) and CS displayed rapid
dissolution in the first 20 days, followed by a remarkably slow
dissolution rate to reach weight retention plateaus of around
13.5%.+-.0.7% and 40.5%.+-.1.4%, respectively. XRD analysis of the
residual from the in vitro dissolution test (FIG. 4b) revealed a
characteristic pattern of poor crystalline HAp for both specimens,
but there were no detectable peaks indicating the existence of CSs.
This indicates that both CS and .alpha.-CSH/ACP (60/40) composites
were transformed to poor crystalline HAp after dissolution in
PBS.
Example 3
Animal Study
Eight beagles (1 year old) were used in this experiment. These dogs
were bred and raised in Ping Tung National University of Science
and Technology. Animal selection, management, and surgical
procedures were approved by the Animal Care and Use Committee of
Taipei Medical University. General and local anesthesia were
performed by an intramuscular injection of 0.1 mg/kg of atropine
(Tai-Yu Co., Hsinchu, Taiwan) and 6-12 mg/kg of Zoletil/Virbac 50
(Virbac Laboratories, France), respectively. Local anesthesia was
performed by injection of lidocaine/epinephrine (Ora Inj.
cartridge, 2% lidocaine hydrochloride and epinephrine 1:73,000,
Showa Yakuhin-Kako, Japan). Extraction of the bilateral mandibular
first molars, and second, third, and fourth premolars (M1 and
P2-P4) was performed in all dogs, and the wounds were allowed to
heal for 3 months. Postoperatively, the dogs were given ampicillin
for an anti-inflammatory effect. Defects of 5- and 8-mm depths were
created by a trephine bur on both sides of the mandible. Bone
defects were filled with .alpha.-CSH/ACP (60/40), Osteoset.RTM.
(CS, Wright Medical Technology, Arlington, Tenn., USA), and an
empty defect was used as a control. Samples (n=4) were harvested
after 3 and 6 weeks. At each harvesting time point, a trephine bur
(6 mm in diameter) was used to collect the specimens, which were
immediately placed in 10% formalin. The sectioned samples were
stained using hematoxylin and eosin. Sections were examined for
evidence of biocompatibility and bone regeneration under a light
microscope. The percentage area of new bone formation was
calculated using the ImageJ 1.37c software (National Institutes of
Health (NIH), Bethesda, Md., USA). The difference of percentage
areas of new bone formed between the treated defects and the
control defect were tested with an unpaired Student's t-test. A p
value of <0.05 indicated statistical significance.
FIG. 5 shows that the empty control group was dominated by
connective tissue. In the Osteoset.RTM. and .alpha.-CSH/ACP groups,
both of which showed obvious bone formation after 3 weeks, some
connective tissue still existed in both groups. Six weeks
postoperatively, no fibrous tissues or inflammatory cells were
detected. Some new bone was observed in the empty control group,
but numerous unhealed cavities were observed. In the Osteoset.RTM.
and .alpha.-CSH/ACP (60/40) groups, the new bone that formed was
very notable, but there were more unhealed cavities in the
Osteoset.RTM. group than in the .alpha.-CSH/ACP (60/40) group. The
exact area of new bone formation was estimated by a
histomorphometric method in the following experiment.
Example 4
Histomorphometry Assay (Quantitative Evaluation)
New bone formation was quantified by image analysis; the results
are summarized in table 1. At 3 weeks, the ratio of new bone
formation for defects treated with .alpha.-CSH/ACP (60/40)
(21.1%.+-.15.0%) was significantly higher than that of the empty
control (13.6%.+-.9.5%) (p<0.05), but there was no significant
difference in the amount of new bone formation between defects
grafted with Osteoset.RTM. (29.0%.+-.16.0%). Thus, filling defects
with .alpha.-CSH/ACP or Osteoset.RTM. enhanced bone regeneration
rates within 3 weeks. Six weeks postoperatively, more bone had
formed in defects treated with .alpha.-CSH/ACP (60/40)
(62.2%.+-.6.8%) than Osteoset.RTM. (53.3%.+-.4.6%) (p<0.01) and
the empty control (40.1%.+-.7.2%) (p<0.0002). The above results
demonstrate the .alpha.-CSH/ACP (60/40) groups showed better
performance; for this reason, adding ACP to CS enhanced the
performance of bone regeneration, especially in the middle stage of
healing.
Finally, the linear least squares fitting technique was used to
estimate the complete healing period for bone defects in the canine
model treated with .alpha.-CSH/ACP (60/40) bone substitutes.
The extrapolation graph (FIG. 6) approximated that the complete
healing period for the .alpha.-CSH/ACP (60/40) bone substitute
would be within a range of 10-12 weeks (10.3 weeks). The usual
canine model has a bone regeneration rate 1.5-2 times faster than
humans, so the 10-12-week healing period in our study may be
equivalent 15-18 weeks (3.75-4.5 months) in humans.
TABLE-US-00002 TABLE 1 New bone formations (%) Groups 3 weeks (SD)
6 weeks (SD) .alpha.-CSH/ACP 21.1 (15.0) * 62.2 (6.8) *** Osteoset
.RTM. 29.0 (16.0) * 53.3 (4.6) * Empty Control 13.6 (9.5) 40.1
(7.2)
* * * * *